Insight into Pseudomonas aeruginosa pyocyanin production under low-shear modeled microgravity.

Long-term space flight impairs the immune system of astronauts, rendering them vulnerable to opportunistic infections. Pseudomonas aeruginosa causes opportunistic infections, particularly in individuals with a compromised immune system; it can be a major health hazard for astronauts during space flight missions. Hence, the production of the most abundant redox active virulence factor, pyocyanin by P. aeruginosa, was assessed under low-shear modeled microgravity (LSMMG) conditions, simulated using a high aspect ratio vessel. Moreover, we evaluated changes in the expression of genes involved in pyocyanin biosynthesis and genes involved in the MexGHI-OpmD operon quorum sensing. Extracellular DNA and H2O2 production were measured, and their correlation with pyocyanin production was examined. Interestingly, the pyocyanin quantity was 2.58-fold lower in the LSMMG conditions compared to the normal gravity. LSMMG caused downregulation of the genes associated with pyocyanin biosynthesis. Interestingly, extracellular DNA and H2O2 release were significantly high in the normal gravity environment. Scanning electron microscopy revealed aggregation and elongated cells under LSMMG. Taken together, these findings suggest that LSMMG did not induce pyocyanin secretion in P. aeruginosa.

Modulatory effect of low-shear modeled microgravity on stress resistance, membrane lipid composition, virulence, and relevant gene expression in the food-borne pathogen Listeria monocytogenes.

The present study investigated the influence of low-shear modeled microgravity (LSMMG) conditions on Listeria monocytogenes stress response (heat, cold, and acid), membrane fatty acid composition, and virulence potential as well as stress-/virulence-associated gene expression. The results showed that LSMMG-cultivated cells had lower survival rate and lower D-values under heat and acid stress conditions compared to cells grown under normal gravity (NG). Interestingly, the cold resistance was elevated in cells cultivated under LSMMG conditions when compared to NG conditions. A higher amount of anteiso-branched chain fatty acids and lower ratio of iso/anteiso were observed in LSMMG cultured cells, which would contribute to increased membrane fluidity. Under LSMMG conditions, upregulated expression of cold stress-related genes (cspA, cspB, and cspD) was noticed but no change in expression was observed for heat (dnaK, groES, clpC, clpP, and clpE) and acid stress-related genes (sigB). The LSMMG-grown cells showed inferior virulence capacity in terms of infection, cell cycle arrest, and apoptosis induction in Caco-2 cells compared to those grown under NG conditions. Approximately 3.65, 2.13, 4.02, and 2.65-fold downregulation of prfA, hly, inlA, and bsh genes, respectively, in LSMMG-cultured cells might be the reason for reduced virulence. In conclusion, these findings suggest that growth under LSMMG conditions stimulates alterations in L. monocytogenes stress/virulence response, perhaps due to changes in lipid composition and related genes expression.

Changes in color and translucency of porcelain-repairing resin composites after thermocycling.

The objective of this study was to determine the changes in color and translucency of dental porcelain-repairing resin composites compared to dental porcelain after thermocycling. Color and spectral reflectance of three shades (A2, A3, and A3.5) of one brand of dental porcelain and three basic shades (A2, A3, and A3.5) and three combinations (A2/A3, A3/3.5, and A2/A3.5) of three brands of porcelain-repairing resin composites (ABT, FSP, and TCR) were measured, before and after thermocycling for 3000 cycles, relative to the illuminant D65. The specimen was 2 mm in thickness, and 1 mm of each shade was layered to make combined shades. Changes in color (DeltaE*ab) and translucency parameter (DeltaTP) were calculated. A general linear model by the material (porcelain or resin composite) and shade was used to compare differences (alpha = 0.05). The range of color changes was 0.68-1.67 in porcelain, 0.56-1.30 in ABT, 2.28-3.10 in FSP, and 0.36-1.15 in TCR. The range of DeltaTP was 0.45-0.96 in porcelain, -0.48 to 0.94 in ABT, -1.31 to 0.82 in FSP, and -0.51 to 1.91 in TCR. After thermocycling, changes in color and TP were correlated with the shade of the material, but not with the material. The discrepancy in the changes of color and translucency after thermocycling between porcelain and porcelain-repairing resin composites should be considered when selecting repairing materials.

Diverse vacA allelic types of Helicobacter pylori in Korea and clinical correlation.

Helicobacter pylori has a diversity of vacA allelic types. The purpose of this study was to correlate the vacA status and the clinical outcome. After constructing specific primers for the vacA signal sequence, H. pylori-positive antral biopsy specimens were examined for the vacA status in 25 gastric ulcers, 31 duodenal ulcers, 22 gastric cancers, 42 chronic gastritis, and 8 gastroduodenal ulcers. The relationship between the vacA allele and the clinical disease was examined. The vacA genotype s1c/m1 is predominant in Korea (71/128, 55.5%). Other strains including s1b or s2 were not found in this study. s1c/m1 was more prominent in duodenal ulcers, than in gastric ulcers (p=0.041) and cancer (p=0.029). Seven out of 8 patients with gastric and coexistent duodenal ulcers had the s1c/m1 allele. No statistical differences in the positive rates of the s1a/m1, s1a/m2, and s1c/m2 alleles among the disease groups were found. In conclusion, s1c/m1 is the main vacA allele in Korea and it is particularly associated with duodenal ulcers.

Identification of Helicobacter pylori in Gallstone, Bile, and Other Hepatobiliary Tissues of Patients with Cholecystitis.

BACKGROUND/AIMS: Bacterial infection is accepted as a precipitating factor in cholesterol gallstone formation, and recent studies have revealed the presence of Helicobacter species in the hepatobiliary system. We utilized the polymerase chain reaction (PCR) to establish the presence of bacterial DNA, including from Helicobacter species, in gallstones, bile juice, and gallbladder mucosa from patients with gallstones. METHODS: At cholecystectomy, 58 gallstones, 48 bile samples, and 46 gallbladder mucosa specimens were obtained and subjected to nested PCR using specific 16S rRNA primers of H. pylori and other bacteria. Bacterial species were identified by DNA sequencing analysis. Bacterial 16S rRNA was detected in 25 out of 36 mixed-cholesterol gallstones, 1 out of 10 pure-cholesterol gallstones, and 9 out of 12 pigmented stones. Furthermore, 16S rDNA sequencing identified Escherichia coli, Pseudomonas, Citrobacter, Klebsiella, and Helicobacter species. RESULTS: Helicobacter DNA was detected in 4 out of 58 gallstones, 6 out of 48 bile samples, and 5 out of 46 gallbladder specimens. Direct sequencing of Helicobacter amplicons confirmed strains of H. pylori in all four gallstones, five out of six bile samples, and three out of five gallbladder specimens. Almost all mixed-cholesterol gallstones appear to harbor bacterial DNA, predominantly E. coli. CONCLUSIONS: H. pylori was also found in the biliary system, suggesting that these bacteria are of etiological importance in gallstone formation.

Evaluation of antagonistic activities of Bacillus subtilis and Bacillus licheniformis against wood-staining fungi: in vitro and in vivo experiments.

The antifungal activity of bacterial strains Bacillus subtilis EF 617317 and B. licheniformis EF 617325 was demonstrated against sapstaining fungal cultures Ophiostoma flexuosum, O. tetropii, O. polonicum, and O. ips in both in vitro and in vivo conditions. The crude active supernatant fractions of 7 days old B. subtilis and B. licheniformis cultures inhibited the growth of sapstaining fungi in laboratory experiments. Thermostability and pH stability of crude supernatants were determined by series of experiments. FT-IR analysis was performed to confirm the surface structural groups of lipoproteins present in the crude active supernatant. Partial purification of lipopeptides present in the crude supernatant was done by using Cellulose anion exchange chromatography and followed by Sephadex gel filtration chromatography. Partially purified compounds significantly inhibited the sapstaining fungal growth by in vitro analysis. The lipopeptides responsible for antifungal activity were identified by electrospray ionization mass spectrometry after partial purification by ion exchange and gel filtration chromatography. Four major ion peaks were identified as m/z 1023, 1038, 1060, and 1081 in B. licheniformis and 3 major ion peaks were identified as m/z 1036, 1058, and 1090 in B. subtilis. In conclusion, the partially purified lipopeptides may belong to surfactin and iturin family. In vivo analysis for antifungal activity of lipopeptides on wood was conducted in laboratory. In addition, the potential of extracts for fungal inhibition on surface and internal part of wood samples were analyzed by scanning electron microscopy.

Changes in surface characteristics of dental resin composites after polishing.

The objectives of this study were (1) to determine in vitro changes in surface roughness and color of dental resin composites after application of three finishing and polishing systems; (2) to evaluate the difference in color stability after immersion in a dye solution after polishing; and (3) to evaluate the effects of surface condition, especially roughness, on measured color depending on the color measuring geometries of specular component excluded (SCE) and specular component included (SCI). Color and surface roughness (R(a)) of resin composites of four brands of A2 shade and one brand of Yellow Enamel shade were measured after polymerization, after polishing with Enhance (Dentsply), Sof-Lex (3M ESPE), or Super-Snap (Shofu) composite finishing and polishing systems. Color was also measured after immersion in 2% methylene blue solution. Color was measured according to the CIELAB color scale. Color changes (DeltaE*(ab)) after polishing/staining and by the measuring geometry were calculated by the equation; DeltaE*(ab) = [(DeltaL*)(2) + (Deltaa*)(2) + (Deltab*)(2)](1/2). Ra value was measured with a surface roughness tester. DeltaE*(ab) and DeltaL* values after polishing and after staining varied among polishing systems when measured with SCE geometry. Composites polished with Super-Snap and Sof-Lex systems showed higher DeltaE*(ab) and DeltaL* values than those polished with Enhance polishing system with SCE geometry. DeltaE*(ab) and DeltaL* values between specimens with different surface conditions measured with SCE geometry were significantly higher than those with SCI (p < 0.01). Changes in R(a) value after polishing was insignificant in most cases.

Genotypes of the Helicobacter pylori vacA signal sequence differ with age in Korea.

BACKGROUND: Studies on Helicobacter pylori genotypes have focused on adults in developed countries, and data on the genotypes of Helicobacter pylori recovered from the children are rare. MATERIALS AND METHODS: One hundred and twenty-eight biopsy samples from patients with H. pylori infection were studied. The patients' ages ranged from 9 to 83 years. PCR analysis for vacA genotypes was performed using DNA extracted from biopsy specimens. RESULTS: Genotyping of the s-region showed s1a in 33 (25.8%) samples and s1c in 82 (64.1%) samples. When the specimens were grouped by age, the distribution of s-region genotype was found to be significantly different between groups (p = .002). The prevalence of s1a was 45.2% in patients < 20 years old, but 14.9% in patients > or = 50 years old. On the other hand, the prevalence of s1c or recombinant s1a-s1c was higher in those > or = 50 years old. The distribution of the m-region did not differ significantly with age (p = .110). CONCLUSIONS: Strain populations infecting Korean adults and children differ.

Effective transduction of osteogenic sarcoma cells by a baculovirus vector.

Efficient gene delivery of a baculovirus-derived vector (BV-p53-lacZ) to a human osteogenic sarcoma cell line, Saos-2, was serendipitously found while evaluating the vector for gene delivery to human p53-null tumour cells in a previous study. Therefore, we investigated other human, rat and mouse osteogenic sarcoma and other types of tumour cell lines for transduction efficiency via baculovirus vectors containing a lacZ reporter gene under the control of either a cytomegalovirus or Rous sarcoma virus promoter. The expression of beta-galactosidase protein, assessed by X-Gal staining and beta-galactosidase ELISA, demonstrated an extremely high level of transduction efficiency in some osteogenic sarcoma cell lines, such as U-2OS, Saos-2 and Saos-LM2. These human osteogenic sarcoma cell lines showed levels of beta-galactosidase expression 5-40 times greater than HepG2 cells, which were previously thought to be the mammalian cells most susceptible to baculovirus-mediated gene delivery. The level of acetylated histone proteins in these tumour lines did not correlate well with the high level of reporter gene expression. These results strongly suggest that some osteogenic sarcoma cells are highly susceptible to baculovirus-mediated gene delivery and that a baculovirus-derived vector is an efficient gene delivery vehicle into human osteogenic sarcoma cells.